As urgently needed vaccines against congenital human cytomegalovirus (HCMV) infection move forward in clinical trials, there remains an incomplete understanding of the optimal vaccination strategy for protection of the newborn. Protein subunit vaccines based on the immunodominant envelope glycoprotein, glycoprotein B (gB), are safe, and induce robust neutralizing antibody titers. Vectored vaccines directed against the major cytotoxic-T-lymphocyte target, the UL83 (pp65) protein, are also under study. However, concerns remain about whether immune responses against HCMV protein subunits alone would be capable of conferring adequate immune responses for protection against congenital HCMV infection and disease, given the diversity of gB coding sequences among clinical isolates and given the large number of other immunogenic CMV proteins. Live, attenuated HCMV vaccines are an attractive alternative, since such vaccines offer the possibility of inducing long-lasting responses to a broad range of viral proteins. The major limitation of live virus vaccines is the inability to ensure adequate safety against latency, reactivation, and transplacental transmission. Based on the hypothesis that live virus vaccines can be engineered for improved safety by deletion of viral immunomodulatory genes, this proposal aims to examine the role of three viral gene families in live virus vaccine-mediated protection and transplacental viral transmission in the only small animal model of congenital CMV infection, the guinea pig model Specifically, we will be investigating a rational live-virus vaccine design strategy based on the hypothesis that deletion of virally-encoded immunoevasive genes will result in an attenuation phenotype, with decreased transplacental transmission, compared to wild-type virus. We further hypothesize that such vaccine candidates will retain immunogenicity and confer protection against congenital GPCMV transmission and disease, while having a better safety profile. The specific viral genes to be evaluated will be: 1) homologs of the viral G-protein coupled receptors (GPCRs), UL33 and UL78; 2) a novel CC Beta chemokine, GPCMV MIP-1; 3) the GPCMV class I down-regulation gene(s). Molecular cloning techniques, including mutagenesis of the GPCMV genome cloned as a bacterial artificial chromosome (BAC), will be utilized to engineer these mutant GPCMV live attenuated vaccine candidates. Vaccine efficacy studies of the live, attenuated viruses will be performed in parallel with pathogenesis analyses, to assess the safety profile of the candidate vaccines (placental infection and injury, transplacental transmission) compared to wild-type GPCMV. Additionally, a signature-tagged mutagenesis strategy will be employed to generate recombinant viruses for in vivo experiments to elucidate the role of other viral genes in congenital transmission and pathogenesis. This will represent the first application of signature tag methodology to the study of congenital infection. The results of these investigations will expand our knowledge of immune evasion strategies employed by the CMVs, will aid understanding of the viral factors responsible for the pathogenesis of congenital CMV disease, and will enable development of rational live-virus HCMV vaccines for human clinical trials.